Identification of muscle necrosis in the mdx mouse model of Duchenne muscular dystrophy using three-dimensional optical coherence tomography

Three-dimensional optical coherence tomography (3D-OCT) was used to image the structure and pathology of skeletal muscle tissue from the treadmill-exercised mdx mouse model of human Duchenne muscular dystrophy. Optical coherence tomography (OCT) images of excised muscle samples were compared with co-registered hematoxylin and eosin-stained and Evans blue dye fluorescence histology. We show, for the first time, structural 3D-OCT images of skeletal muscle dystropathology well correlated with co-located histology. OCT could identify morphological features of interest and necrotic lesions within the muscle tissue samples based on intrinsic optical contrast. These findings demonstrate the utility of 3D-OCT for the evaluation of small-animal skeletal muscle morphology and pathology, particularly for studies of mouse models of muscular dystrophy.     

Contribution of oxidative stress to pathology in diaphragm and limb muscles with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease that makes walking and breathing difficult. DMD is caused by an X-linked (Xp21) mutation in the dystrophin gene. Dystrophin is a scaffolding protein located in the sarcolemmal cytoskeleton, important in maintaining structural integrity and regulating muscle cell (muscle fiber) growth and repair. Dystrophin deficiency in mouse models (e.g., mdx mouse) destabilizes the interface between muscle fibers and the extracellular matrix, resulting in profound damage, inflammation, and weakness in diaphragm and limb muscles. While the link between dystrophin deficiency with inflammation and pathology is multi-factorial, elevated oxidative stress has been proposed as a central mediator. Unfortunately, the use of non-specific antioxidant scavengers in mouse and human studies has led to inconsistent results, obscuring our understanding of the importance of redox signaling in pathology of muscular dystrophy. However, recent studies with more mechanistic approaches in mdx mice suggest that NAD(P)H oxidase and nuclear factor-kappaB are important in amplifying dystrophin-deficient muscle pathology. Therefore, more targeted antioxidant therapeutics may ameliorate damage and weakness in human population, thus promoting better muscle function and quality of life. This review will focus upon the pathobiology of dystrophin deficiency in diaphragm and limb muscle primarily in mouse models, with a rationale for development of targeted therapeutic antioxidants in DMD patients.     

A Neurogenic Component in Muscular Dystrophy

Evidence for a neurogenic component in mouse and human muscular dystrophy is briefly reviewed. Such evidence comes from certain clinical observations, electrophysiological studies, muscle pathology, nervous system pathology, transplantation experiments in animals, and tissue culture studies. The evidence is at present rather conflicting though the results of recent tissue culture experiments are more convincing. If there is a neurogenic component in dystrophy then the basic defect may have to be sought in the central nervous system rather than in the muscle itself. It is argued, however, that a neurogenic component in dystrophy cannot be simply a defect in the anterior horn cells of the spinal cord since the clinical features and the laboratory and pathological findings are quite different from those in spinal muscular atrophy.     

Preservation of muscle force in Mdx3cv mice correlates with low-level expression of a near full-length dystrophin protein

The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at approximately 5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients.     

Hearing loss in the laminin-deficient dy mouse model of congenital muscular dystrophy

Sensorineural hearing loss is found in many inherited forms of muscular dystrophy. We investigated the dy mouse model, which has congenital muscular dystrophy due to a defect in laminin alpha 2, for evidence of cochlear dysfunction. Auditory brainstem response (ABR) audiometry to pure tones was used to evaluate 3-month-old homozygous dy/dy and age-matched C57 control mice. The average ABR thresholds to tone-burst stimuli for four frequencies (4, 8, 16, and 32 kHz) were determined and statistically compared by ANOVA. The dy/dy mice demonstrated elevated auditory thresholds ranging from 25 to 27 dB at each frequency tested (p<0.0001). Anatomic evaluations of the ears revealed pathology ranging from extensive connective tissue infiltration within the inner ear to possible minor defects in the cells of the organ of Corti. These anatomic and physiologic observations suggest that the extracellular matrix protein laminin plays a crucial role in normal cochlear function. Furthermore, the dy congenital muscular dystrophy mouse offers a novel model for evaluation of sensorineural hearing loss associated with muscular dystrophy.     

Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype

M1 macrophages play a major role in worsening muscle injury in the mdx mouse model of Duchenne muscular dystrophy. However, mdx muscle also contains M2c macrophages that can promote tissue repair, indicating that factors regulating the balance between M1 and M2c phenotypes could influence the severity of the disease. Because interleukin-10 (IL-10) modulates macrophage activation in vitro and its expression is elevated in mdx muscles, we tested whether IL-10 influenced the macrophage phenotype in mdx muscle and whether changes in IL-10 expression affected the pathology of muscular dystrophy. Ablation of IL-10 expression in mdx mice increased muscle damage in vivo and reduced mouse strength. Treating mdx muscle macrophages with IL-10 reduced activation of the M1 phenotype, assessed by iNOS expression, and macrophages from IL-10 null mutant mice were more cytolytic than macrophages isolated from wild-type mice. Our data also showed that muscle cells in mdx muscle expressed the IL-10 receptor, suggesting that IL-10 could have direct effects on muscle cells. We assayed whether ablation of IL-10 in mdx mice affected satellite cell numbers, using Pax7 expression as an index, but found no effect. However, IL-10 mutation significantly increased myogenin expression in vivo during the acute and the regenerative phase of mdx pathology. Together, the results show that IL-10 plays a significant regulatory role in muscular dystrophy that may be caused by reducing M1 macrophage activation and cytotoxicity, increasing M2c macrophage activation and modulating muscle differentiation.     

Major basic protein-1 promotes fibrosis of dystrophic muscle and attenuates the cellular immune response in muscular dystrophy

The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.     

Age-dependent effect of myostatin blockade on disease severity in a murine model of limb-girdle muscular dystrophy

Myostatin (MSTN) is a muscle-specific secreted peptide that functions to limit muscle growth through an autocrine regulatory feedback loop. Loss of MSTN activity in cattle, mice, and humans leads to a profound phenotype of muscle overgrowth, associated with more and larger fibers and enhanced regenerative capacity. Deletion of MSTN in the mdx mouse model of Duchenne muscular dystrophy enhances muscle mass and reduces disease severity. In contrast, loss of MSTN activity in the dyW/dyW mouse model of laminin-deficient congenital muscular dystrophy, a much more severe and lethal disease model, does not improve all aspects of muscle pathology. Here we examined disease severity associated with myostatin (mstn-/-) deletion in mice nullizygous for delta-sarcoglycan (scgd-/-), a model of limb-girdle muscular dystrophy. Early loss of MSTN activity achieved either by monoclonal antibody administration or by gene deletion each improved muscle mass, regeneration, and reduced fibrosis in scgd-/- mice. However, antibody-mediated inhibition of MSTN in late-stage dystrophic scgd-/- mice did not improve disease. These findings suggest that MSTN inhibition may benefit muscular dystrophy when instituted early or if disease is relatively mild but that MSTN inhibition in severely affected or late-stage disease may be ineffective.     

ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice

Muscular dystrophy is characterized by muscle degeneration and insufficient regeneration and replacement of muscle fibers by connective tissue. New therapeutic strategies directed toward various forms of muscular dystrophy are needed to preserve muscle mass and promote regeneration. In this study we examined the role of the transmembrane ADAM12, a disintegrin and metalloprotease, which is normally associated with development and regeneration of skeletal muscle. We demonstrate that ADAM12 overexpression in the dystrophin-deficient mdx mice alleviated the muscle pathology in these animals, as evidenced by less muscle cell necrosis and inflammation, lower levels of serum creatine kinase, and less uptake of Evans Blue dye into muscle fibers. These studies demonstrate that ADAM12 directly or indirectly contributes to muscle cell regeneration, stability, and survival.     

Combined deficiency of alpha and epsilon sarcoglycan disrupts the cardiac dystrophin complex

Cardiomyopathy is a puzzling complication in addition to skeletal muscle pathology for patients with mutations in β-, γ- or δ-sarcoglycan (SG) genes. Patients with mutations in α-SG rarely have associated cardiomyopathy, or their cardiac pathology is very mild. We hypothesize that a fifth SG, ε-SG, may compensate for α-SG deficiency in the heart. To investigate the function of ε-SG in striated muscle, we generated an Sgce-null mouse and a Sgca-;Sgce-null mouse, which lacks both α- and ε-SGs. While Sgce-null mice showed a wild-type phenotype, with no signs of muscular dystrophy or heart disease, the Sgca-;Sgce-null mouse developed a progressive muscular dystrophy and a more anticipated and severe cardiomyopathy. It shows a complete loss of residual SGs and a strong reduction in both dystrophin and dystroglycan. Our data indicate that ε-SG is important in preventing cardiomyopathy in α-SG deficiency.     

Coelomocytes as Effector Cells in Eartworm Immunity

The results from this study support the following hypothetical mechanism for the rejection of xenografts by Lumbricus. Initially, coelomocytes are attracted to xenografts due to the stimulus of tissue injury. This results in clumping or nodule formation around injured and loose muscle fibers, and a general clumping of baso-phils. Clumping may entran acidophilic cells and/or attract such cells to the graft site. Because the graft is a foreign tissue, its foreignness also stimulates a cellular response. Basophils invade the muscular structure of the xenograft and more acidophilic cells accumulate at the graft site. The acidophilic cells have the canacity to digest the connective tissue elements which hold the muscle fibers of the graft in place. Dissolution of these connective tissue elements results in the release of muscle fibers. The presence of free foreign muscular tissue in the coelomic cavity acts as a stimulus for further accumulations of basophilic cells, which in turn form more capsules and may attract more acidophils. Thus there is a continuous erosion of xenograft muscle by acidophils and a clearing away of debris by basophils. This process eventually leads to the complete destruction of a xenograft.

Autografts stimulate the accumulation of basophils due to the presence of injured tissue. The presence of cell clumps and/or injured tissue also causes acidophilic cells to accumulate under the graft. Only injured or dead tissue is acted upon by the coelomic cells since the stimulation of any further cellular invasion into the graft by a foreign antigen, is not present in autografts. Acidophilic coelomic cells, either do not release substances that can attack the connective tissues of autografts, or the substances released by these cells does not react with non-injured self tissue. This mechanism together with other information concerning specificity and anamnesis in the rejection response of the annelid Lumbricus terrestris, offers further support for a true immunologi-cal response to tissue grafts in an invertebrate.     

Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal, muscle-wasting disease of childhood. Previous investigations have shown that muscle macrophages may play an important role in promoting the pathology in the mdx mouse model of DMD. In the present study, we investigate the mechanism through which macrophages promote mdx dystrophy and assess whether the phenotype of the macrophages changes between the stage of peak muscle necrosis (4 weeks of age) and muscle regeneration (12 weeks). We find that 4-week-old mdx muscles contain a population of pro-inflammatory, classically activated M1 macrophages that lyse muscle in vitro by NO-mediated mechanisms. Genetic ablation of the iNOS gene in mdx mice also significantly reduces muscle membrane lysis in 4-week-old mdx mice in vivo. However, 4-week mdx muscles also contain a population of alternatively activated, M2a macrophages that express arginase. In vitro assays show that M2a macrophages reduce lysis of muscle cells by M1 macrophages through the competition of arginase in M2a cells with iNOS in M1 cells for their common, enzymatic substrate, arginine. During the transition from the acute peak of mdx pathology to the regenerative stage, expression of IL-4 and IL-10 increases, either of which can deactivate the M1 phenotype and promote activation of a CD163+, M2c phenotype that can increase tissue repair. Our findings further show that IL-10 stimulation of macrophages activates their ability to promote satellite cell proliferation. Deactivation of the M1 phenotype is also associated with a reduced expression of iNOS, IL-6, MCP-1 and IP-10. Thus, these results show that distinct subpopulations of macrophages can promote muscle injury or repair in muscular dystrophy, and that therapeutic interventions that affect the balance between M1 and M2 macrophage populations may influence the course of muscular dystrophy.     

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans,mice, and zebrafish

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.     

Coelomocytes as Effector Cells in Eartworm Immunity

The results from this study support the following hypothetical mechanism for the rejection of xenografts by Lumbricus. Initially, coelomocytes are attracted to xenografts due to the stimulus of tissue injury. This results in clumping or nodule formation around injured and loose muscle fibers, and a general clumping of baso-phils. Clumping may entran acidophilic cells and/or attract such cells to the graft site. Because the graft is a foreign tissue, its foreignness also stimulates a cellular response. Basophils invade the muscular structure of the xenograft and more acidophilic cells accumulate at the graft site. The acidophilic cells have the canacity to digest the connective tissue elements which hold the muscle fibers of the graft in place. Dissolution of these connective tissue elements results in the release of muscle fibers. The presence of free foreign muscular tissue in the coelomic cavity acts as a stimulus for further accumulations of basophilic cells, which in turn form more capsules and may attract more acidophils. Thus there is a continuous erosion of xenograft muscle by acidophils and a clearing away of debris by basophils. This process eventually leads to the complete destruction of a xenograft.

Autografts stimulate the accumulation of basophils due to the presence of injured tissue. The presence of cell clumps and/or injured tissue also causes acidophilic cells to accumulate under the graft. Only injured or dead tissue is acted upon by the coelomic cells since the stimulation of any further cellular invasion into the graft by a foreign antigen, is not present in autografts. Acidophilic coelomic cells, either do not release substances that can attack the connective tissues of autografts, or the substances released by these cells does not react with non-injured self tissue. This mechanism together with other information concerning specificity and anamnesis in the rejection response of the annelid Lumbricus terrestris, offers further support for a true immunologi-cal response to tissue grafts in an invertebrate.     

Transgenic overexpression of ADAM12 suppresses muscle regeneration and aggravates dystrophy in aged mdx mice

Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy.     

Transgenic overexpression of dystroglycan does not inhibit muscular dystrophy in mdx mice

Recently, there have been a number of studies demonstrating that overexpression of molecules in skeletal muscle can inhibit or ameliorate aspects of muscular dystrophy in the mdx mouse, a model for Duchenne muscular dystrophy. Several such studies involve molecules that increase the expression of dystroglycan, an important component of the dystrophin-glycoprotein complex. To test whether dystroglycan itself inhibits muscular dystrophy in mdx mice, we created dystroglycan transgenic mdx mice (DG/mdx). The alpha and beta chains of dystroglycan were highly overexpressed along the sarcolemmal membrane in most DG/mdx muscles. Increased dystroglycan expression, however, did not correlate with increased expression of utrophin or sarcoglycans, but rather caused their decreased expression. In addition, the percentage of centrally located myofiber nuclei and the level of serum creatine kinase activity were not decreased in DG/mdx mice relative to mdx animals. Therefore, dystroglycan overexpression does not cause the concomitant overexpression of a utrophin-glycoprotein complex in mdx muscles and has no effect on the development of muscle pathology associated with muscular dystrophy.     

Resolution of skeletal muscle inflammation in mdx dystrophic mouse is accompanied by increased immunoglobulin and interferon-gamma production

Mdx mouse, the animal model of Duchenne muscular dystrophy, develops an X-linked recessive inflammatory myopathy with an apparent sustained capacity for muscle regeneration. We analysed whether changes in the skeletal muscle during myonecrosis and regeneration would correlate with functional alterations in peripheral lymphoid tissues. Here we show that during the height of myonecrosis, mdx mice display marked atrophy of peripheral lymph nodes and extensive muscle inflammation. In contrast, enlargement of draining lymph nodes with accumulation of CD4+ CD44+, CD4+ CD25+, CD8+ CD44+ T lymphocytes and type-2 B cells was consistently observed during amelioration of the muscle lesion. In addition, regeneration of the muscular tissue was accompanied by concomitant increase of immunoglobulin-secreting cells in regional lymph nodes and bone marrow. Double immunolabelling analysis revealed intense B cell proliferation and formation of germinal centre in the follicles of dystrophic regional lymph nodes. Furthermore, lymph node cells produced large amounts of IFN-gamma but not IL-4, IL-6 or IL-10 after in vitro mitogen stimulation with Concanavalin A. As these alterations occurred mainly during the recovery period, we suggested that local activation of the immune system could be an influence which mitigates the myonecrosis of muscular tissue in the mdx dystrophic mouse.     

Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.     

Overexpression of the cytotoxic T cell (CT) carbohydrate inhibits muscular dystrophy in the dyW mouse model of congenital muscular dystrophy 1A

A number of recent studies have demonstrated therapeutic effects of transgenes on the development of muscle pathology in the mdx mouse model for Duchenne muscular dystrophy, but none have been shown also to be effective in mouse models for laminin alpha2-deficient congenital muscular dystrophy (MDC1A). Here, we show that overexpression of the cytotoxic T cell (CT) GalNAc transferase (Galgt2) is effective in inhibiting the development of muscle pathology in the dy(W) mouse model of MDC1A, much as we had previously shown in mdx animals. Embryonic overexpression of Galgt2 in skeletal muscles using transgenic mice or postnatal overexpression using adeno-associated virus both reduced the extent of muscle pathology in dy(W)/dy(W) skeletal muscle. As with mdx mice, embryonic overexpression of the Galgt2 transgene in dy(W)/dy(W) myofibers inhibited muscle growth, whereas postnatal overexpression did not. Both embryonic and postnatal overexpression of Galgt2 in dy(W)/dy(W) muscle increased the expression of agrin, a protein that, in recombinant form, has been shown to ameliorate disease, whereas laminin alpha1, another disease modifier, was not expressed. Galgt2 over-expression also stimulated the glycosylation of a gly-colipid with the CT carbohydrate, and glycolipids accounted for most of the CT-reactive material in postnatal overexpression experiments. These experiments demonstrate that Galgt2 overexpression is effective in altering disease progression in skeletal muscles of dy(W) mice and should be considered as a therapeutic target in MDC1A.     

Elimination of myostatin does not combat muscular dystrophy in dy mice but increases postnatal lethality

Myostatin is a TGF-beta family member and a negative regulator of skeletal muscle growth. It has been proposed that reduction or elimination of myostatin could be a treatment for degenerative muscle diseases such as muscular dystrophy. Laminin-deficient congenital muscular dystrophy is one of the most severe forms of muscular dystrophy. To test the possibility of ameliorating the dystrophic phenotype in laminin deficiency by eliminating myostatin, we crossed dy(W) laminin alpha2-deficient and myostatin null mice. The resulting double-deficient dy(W)/dy(W);Mstn(-/-) mice had a severe clinical phenotype similar to that of dy(W)/dy(W) mice, even though muscle regeneration was increased. Degeneration and inflammation of muscle were not alleviated. The pre-weaning mortality of dy(W)/dy(W);Mstn(-/-) mice was increased compared to dy(W)/dy(W), most likely due to significantly less brown and white fat in the absence of myostatin, and postweaning mortality was not significantly improved. These results show that eliminating myostatin in laminin-deficiency promotes muscle formation, but at the expense of fat formation, and does not reduce muscle pathology. Any future therapy based on myostatin may have undesirable side effects.